An inflammatory response is local response to cellular injury that is marked by capillary dilation, leukocytic infiltration, redness, heat, and pain and serves as a mechanism initiating the elimination of noxious agents and of damaged tissue.
A generalized inflammatory response within a tissue occurs by the recruitment of leukocytes to the tissue. Destruction of bacteria, foreign materials and/or damaged cells occurs through phagocytosis and/or extracellular degranulation (secretion of degradative enzymes, antimicrobial proteins and myeloperoxidase, which forms superoxides from secreted H2O2). While most localized inflammatory responses are beneficial, harmful inflammatory responses can occur. Many harmful inflammatory responses also involve accumulation of leukocytes within a tissue. This accumulation results in the destruction of viable cells and tissue. In addition to damaging tissue, these responses are detrimental to, or debilitating for, the afflicted individual. Examples of detrimental inflammatory responses can include the following; ischemia/reperfusion injury following myocardial infarction, shock, stroke, organ transplantation, cardiopulmonary bypass surgery, allograft rejection, rheumatoid arthritis, antigen-induced asthma, allergic rhinitis, and glomerulonephritis (see the review in; Harlan, et al., Immunol. Rev., 114:5-12, 1990; Carlos and Harlan, Blood, 84:2068-2101, 1994.)
Leukocyte recruitment involves a cascade of cellular events, beginning with activation of vascular endothelium by damaged or infected tissue adjacent to the endothelium. Activation of the endothelium results in enhanced adhesion of leukocytes to the endothelial cells, and transendothelial migration (extravasation) by bound leukocytes into the damaged tissue. Endothelial activation is manifested by a short-term, rapid, and/or a long-term stimulation of the endothelial cells.
Activators such as thrombin, chemoattractant leukotrienes, B4, C4 and D4 (LTB4, LTC4 & LTD4), and histamine cause rapid, transient (<30 minutes) endothelial cell activation, independent of protein synthesis, and can increase endothelial cell surface levels of the chemoattractants such as platelet activating factor (PAF; a glycerophospholipid) and LTB4 (shown for histamine), and adhesion molecules; ICAM-1 (shown for thrombin) and P-selectin (Zimmerman, et al., J. Cell Biol., 110:529-540, 1990; Sugama, et al., J. Cell Biol., 119:935-944, 1992; McIntyre, et al. Proc. Natl. Acad. Sci. USA, 83:2204-2208, 1986; Lorant, et al., J. Cell Biol., 115:223-234, 1991). The outcome of rapid activation of endothelial cells is increased leukocyte adhesion to the endothelium (Hoover, et al., Proc. Natl. Acad. Sci. USA, 81:2191-2194, 1984; Zimmerman, et al., J. Cell Biol., 110:529-540, 1990; Hanahan, et al. Ann. Rev. Biochem., 55:483-509, 1986). However, increased LTB4 surface levels have not been shown to directly increase transendothelial migration of neutrophils (Hughes, et al., Prost. Leuk. Essent. F. A., 45:113-119, 1992), and in certain situations, PAF is not necessary for adhesion of leukocytes to activated endothelium (Kuijpers, et al., J. Cell Biol., 117:565-574, 1992).
Long-term (hours in duration) protein synthesis dependent, endothelial cells activation is produced by cytokines, such as IL-1b and TNF-a, and by lipopolysaccharide (LPS) and results in maintenance of increased surface levels of adhesion molecules: E-selectin, P-selectin, ICAM-1 and VCAM-1 (reviewed by Carlos, and Harlan, Blood, 84:2068-2101, 1994). IL-1b and TNF-a also increase the synthesis of PAF by endothelial cells (Kuijpers, et al., J. Cell Biol., 117:565-574, 1992). In addition, endothelial cell activation by IL-1b, TNF-a, LPS and histamine has been shown to increase the synthesis and secretion of the chemokine, IL-8 (Strieter, et al., Science, 243:1467-1469, 1989; Jeannin, et al., Blood, 84:2229-2233, 1994).
Chemokines, IL-8 and MCP-1, have been shown to be produced by and to be present on the endothelial cells surface (Huber, et al., Science, 254:99-102, 1991; Springer, Nature, 346:425-434, 1990). The chemokine, MIP-1b, has been shown to be present on lymph node endothelium, in vivo (Taub, et al., Science, 260:355-359, 1993; Tanaka, et al., Nature, 361:79-82, 1993). The chemokines; RANTES, MIP-a, MIP-b, MCP-1 and IL-8 are all heparin binding proteins, which after being secreted, bind to cell surface and extracellular matrix proteoglycans possessing heparin and heparan sulfate moieties (reviewed by Miller, et al., Crit. Rev. Immunol., 12:17-46, 1992).
Heparin and heparan sulfate are similar glycosaminoglycan moieties found interspersed on the same unbranched carbohydrate chains. They are covalently attached to the protein backbones of proteoglycans. Despite what these two names imply, heparin is more highly sulfated than is heparan sulfate. Proteoglycans are present on cell surfaces and in extracellular matrices (e.g. in the basement membrane of endothelium). Because of difficulty in distinguishing regions of heparin and heparan sulfate on the same carbohydrate chain, little data exists on the binding preference of chemokines for either heparin or heparan sulfate moieties. There is some indication that chemokines IL-8 and GRO bind with greater affinity to heparan sulfate than heparin, and that PF4 and NAP-2 bind with greater affinity to heparin moieties (Witt, and Lander, Curr. Biol., 4:394400, 1994). Generally, chemokines are referred to as heparin binding proteins. C-terminal regions of the chemokines IL-8, PF4, MCP-1 and NAP-2 have been shown to form an a-helix, and to bind to heparin/heparan sulfate (Webb, et al., Proc. Natl. Acad. Sci. USA, 90:7158-7162, 1993; Zucker, et al., Proc. Natl. Acad. Sci. USA, 86:7571-7574, 1989; Matsushima, et al., in Interleukins: Molec. Biol. Immunol., ed. Kistimoto, Karger, Basel, 236-265, 1992). This is likely to be a structure, common to all of the chemokines.
All of the molecules mentioned above, which are expressed by activated endothelial cells (PAF, LTB4, selectins, CAMs and chemokines), are present on the endothelial cell surface, and are localized to vascular endothelium adjacent to sites of damaged tissue. Blood-borne leukocytes which interact with these molecules will also be localized in their binding in the area of the damaged tissue. The outcome of long-term activation of endothelium is increased adhesion and extravasation of leukocytes and a significant localized accumulation of leukocytes in adjacent tissue, which cannot occur during short-term activation (Ebisawa, et al., J. Immunol., 149:4021-4028, 1992; Huber, and Weiss, J. Clin. Invest., 83:1122-1129, 1989; Oppenheimer-Marks, et al., J. Immunol., 145:140-148, 1990).
Adhesion of leukocytes to endothelium is thought to be a two step process (reviewed by Carlos, and Harlan, Blood, 84:2068-2101, 1994). Initially, leukocytes roll along the surface of blood vessels. Increased rolling is initially mediated on vascular endothelium (within the first 30 minutes) by interactions between Sialyl Lewisx structures on the leukocyte surface and P-selectin and E-selectin, which are increased on activated endothelial cells (Ley, et al., Blood, 85:3727-3735, 1995). Increased rolling is also mediated (after 40 minutes) by interactions between L-selectin on leukocyte cellular membranes and heparin-like molecules on the vascular endothelium, which are cytokine-inducable (Karin. et al., Science, 261:480-483, 1993), or between L-selectin on lymphocytes and vascular addressins; GlyCAM-1, CD34 and MAdCAM-1 on high endothelial venules (HEVS) in lymphoid tissue. The second step, firm adhesion of leukocytes to endothelial cells, is based on interactions between leukocyte integrins (e.g. LFA-1, Mac-1, VLA-4) and endothelial cellular adhesion molecules (CAMs; e.g. ICAM-1, ICAM-2, VCAM-1, MAdCAM-1). Leukocytes flatten on the endothelial surface, and shed L-selectin, concomitant with firm adhesion (Kishimoto, et al., Science, 245:1238-1242, 1989; Jutila, et al., J. Immunol., 143:3318-3324, 1989; Smith, et al., Clin. Invest., 87:609-618, 1991).
Selectin and CAM levels increase on the endothelium surface in response to many cytokines and chemoattractants. These increases are dependent on synthesis and/or secretion of additional selectin and CAM molecules onto the cell surface. In contrast, activation of leukocytes for firm adhesion has been shown to occur within second (Bargatze, et al., J. Exp. Med., 178:367-373, 1993), through increased secretion of integrins, and more importantly, through induction of conformational changes in cell surface integrins (integrin activation), which permits tight binding of the integrins to CAMs (reviewed in Zimmerman, Immunol. Today, 13:93-99, 1992).
PAF and E-selectin can activate integrins for endothelial cell adhesion (Lorant et al., J. Biol. Chem., 115:223-234, 1991; Lo, J. Exp. Med., 173:1493-1500, 1991). The presence of MIP-1b, immobilized by binding to CD44 (possesses heparin/heparan sulfate moieties), or a heparin-BSA conjugate, has been shown to be required for CD8+ T-cell binding to immobilized VCAM-1 molecules. This binding was shown to be blocked by an antibody to VLA-4, indicating that MIP-1b activates VLA-4 on the T-cell surface (Tanaka, et al., Nature, 361:79-82, 1993). An increase in the level of integrin, CD18 (part of Mac-1), on the surface of neutrophils has been shown to occur when neutrophils contact endothelium, which has been stimulated with IL-1b. An antibody to IL-8 inhibited the CD18 up-regulation, and also inhibited neutrophil adhesion (Huber, Science, 254:99-102, 1991). Thus, chemokines can act as direct activators of leukocyte adhesion. In contrast, Luscinaskas et al. (J. Immunol., 149:2163-2171, 1992) has demonstrated that pretreatment of neutrophils with IL-8 inhibits neutrophil attachment, and addition of exogenous IL-8 detached neutrophils adhering to activated endothelial cells. Rot (Immunol. Today, 13:291-294, 1992) has reconciled these contradictory results by proposing that IL-8 bound to the endothelial cells surface promotes adhesion, while soluble IL-8 can inhibit it.
Different chemokines activate different leukocytes. IL-8 activates neutrophils, eosinophils and T cells. RANTES activates monocytes, eosinophils and T cells. MCP-1 activates monocytes. MIP-1a activates CD4+ T cells, monocytes and B cells, while MIP-1b activates monocytes and CD8+ T cells (reviewed in Lasky, Current Biol., 3:366-378, 1993). Different combinations of selectins, integrins, CAMs and chemokines are thought to select for the adhesion and migration of the leukocyte subtypes observed in different inflamed tissues (Butcher, Cell, 67:1033-1039, 1991).
The importance of interactions of integrins, CD11/CD18 (Mac-1), and ICAMs in adhesion and extravasation of leukocytes has been demonstrated in numerous systems by the use of antibodies to these molecules. The antibodies interfere with the function of the adhesion molecules and block or reduce leukocyte recruitment. The leukocyte adhesion deficiency (LAD) Type I syndrome results in a partial or total absence of the integrin, CD18, on the leukocytes of affected patients. Neutrophil recruitment to sites of inflammation is negligible. However, monocyte and eosinophil recruitment is normal, indicating that an alternative set of adhesion molecules may function for recruitment of these cells, perhaps VLA-4 and VCAM-1 (Harlan, Clin. Immunol. Immunopath., 67:S16-S24, 1993). VLA-4 is not expressed by neutrophils (Winn and Harlan, J. Clin. Invest., 92:1168-1174, 1993). As mentioned previously, chemokines are important for activation and increased surface levels of integrins VLA-4 and CD18 on leukocytes. IL-8 immobilized on a polycarbonate filter has been shown to be adequate for directing migration of neutrophils through the filter (Rot, Immunol. Today, 13:291-294). Huber, et al. (Science, 254:99-102, 1991) has shown that a transendothelial gradient of bound IL-8, produced by IL-1b stimulated endothelial cell monolayers, is necessary for extravasation of neutrophils. These neutrophils were pre-activated with IL-8 and did bind to the endothelial cells, but did not migrate until the IL-8 gradient was present. This gradient extended from the endothelial cells luminal surface through the basement membrane underlying the endothelial cell monolayer. Washing bound IL-8 from the basement membrane underlying activated endothelial cells prevented migration across the monolayer. In addition, an antibody to IL-8 inhibited 70-80% of the neutrophil migration. Kuijpers et al. (J. Cell Biol., 117:565-572, 1992) used an anti-IL-8 antibody to produce a 60% reduction in neutrophil migration across IL-1b and TNF-a activated endothelium, and addition of a PAF receptor antagonist produced an 85-90% reduction in migration. These results are in contrast to experiments which showed that IL-8 pretreated neutrophils were inhibited in their ability to migrate through an activated endothelial cell monolayer (Luscinaskas et al., J. Immunol., 149:2163-2171, 1992). Thus, it is likely that chemokines not only activate leukocytes for adhesion, but that a bound gradient of chemokine is important in extravasation of leukocytes. The presence of soluble chemokine can interfere with adhesion and migration along bound chemokine gradient. A discussed below, in vivo localized concentration increases in soluble chemokines would be minimized by blood flow.
Once activated leukocytes have begun to accumulate within a damaged tissue, they can augment the accumulation of additional leukocytes, by synthesis and secretion of cytokines, chemokines, and LTB4. LPS has been shown to directly increase monocyte IL-1b expression (Porat, et al., FASEB J., 6:2482-2489, 1992). IL-8, IL-1b and TNF-a are produced by neutrophils activated with GM-CSF, another cytokine produced by activated macrophages, endothelium and T-cells (McCain, et al., Am. J. Respir. Cell Molec. Biol., 8:28-34, 1993; Lindemann, et al., J. Immunol. 140:837-839, 1988; Lindemann, et al., J. Clin. Invest., 83:1308-1312, 1989). IL-1b and TNF-a have been shown to stimulate monocytes, thereby increasing the expression of the chemokines, IL-8 and MIP-1a (Lukacs, et al., Blood, 82:3668-3674, 1993). Activated neutrophils and monocytes have been shown to be a major source of LTB4 production (Samuelsson, et al., Science, 237:1171-1176, 1987; Brach, et al., Eur. J. Immunol., 22:2705-2711, 1992). As discussed previously, LTB4 is not directly involved in further recruitment of leukocytes, but because neutrophils stimulated with LTB4 produce IL-8, the LTB4-stimulated neutrophils could promote further neutrophil recruitment, indirectly, through formation of an IL-8 gradient (McCain, et al., Am. J. Respir. Cell Molec. Biol., 10:651-657, 1994). The continued recruitment of leukocytes by these leukocyte-derived activators would require using the vascular endothelium as an intermediate. Endothelial cells and basement membranes would bind and display neutrophil-derived chemokines, forming a gradient, or leukocyte-derived cytokines would activate the endothelium, which would also cause the creation of a chemokine gradient.
Blood flow in the vasculature would prevent a localized concentration increase in soluble activation factors (cytokines, chemokines and chemoattractants), produced by a tissue-localized inflammatory response. If a local inflammation is producing high blood concentrations of activators, a systemic activation of leukocytes could occur (Finn, et al., J. Thorac. J. Surg., 105:234-241, 1993). The activated leukocytes would then bind transiently to unactivated endothelium and/or degranulate, causing sepsis (Sawyer, et al., Rev. Infect. Dis., 11:S1532-1544, 1989). In situations where some blood-borne leukocytes are activated by a localized inflammation (not all of the activated leukocytes extravasate), the activated leukocytes would produce and secrete additional cytokines, chemokines, and LTB4 into the blood. This increase in activator concentration could up-regulate unactivated cells and amplify the systemic response.
Although the mechanism of inflammatory responses has been given in some detail, there is still a need for an effective treatment and pharmaceutical compositions for reducing or preventing localized inflammatory responses.